3 Биологические мембраны. Обмен веществом (1160072), страница 11
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& Ingraham, J.L. (1962) Effect of temperature on the compositionof fatty acids in Escherichia coli. J. Bacteriol. 84, 1260.* The exact fatty acid composition depends not only on growth temperature, but also on growthstage and growth medium composition.t The total percentage of 16:1 plus 18:1 divided by total percentage of 14:0 plus 16:0.Hydroxymyristic acid was omitted from this calculation.Figure 10-6 Lipid motion within a bilayer includes lateral diffusion of individual moleculeswithin one face of the bilayer, and thermal motionof the fatty acyl groups in the bilayer interior.
Because of the thermal motion, the bilayer is fluidabove a certain temperature; as the temperature islowered, lipids become paracrystalline. Flip-flop diffusion is a very uncommon event.274Part II Structure and CatalysisHOCHoCH.NHIICH3—C—OCH2CH3NHEAC H 3 - C - N H f Protein"NHIICH3—C—OCH2CH2SC>3HOCH2CH2SO.,IEA(a)Although lateral migration of membrane components and thermalflexing of the acyl chains clearly occurs, a third kind of motion is restricted: transbilayer or "flip-flop" diffusion, the movement of a lipidfrom one face of the bilayer to the other (Fig.
10-6). For such motion tooccur, the lipid head group, which is polar and may be charged, mustleave its aqueous environment and move into the hydrophobic interiorof the bilayer, a process with a large, positive free-energy change(highly endergonic). During synthesis of the bacterial plasma membrane, for example, phospholipids produced on the inside surface of themembrane must undergo flip-flop diffusion to enter the outer face ofthe bilayer. There are proteins that facilitate flip-flop diffusion, providing a transmembrane path that is energetically more favorable.Erythrocyte membranecontaining three proteinsMembrane Proteins Penetrate and Span the Lipid BilayerEA labels intracellular andextracellular domains of proteinsIEA labels only extracellulardomains of proteinsMembranes isolated,dissolved in SDS;proteins separated bySDS polyacrylamidegel electrophoresisIEAP2Radioactive proteinsdetected by exposingx-ray film to gel(autoradiography)PiP3(b)When individual protein molecules and multiprotein complexes infreeze-fractured biological membranes are visualized with the electronmicroscope (Box 10-1), some proteins appear on only one face of themembrane; most span the full thickness of the bilayer, and protrudefrom both inner and outer membrane surfaces.
Among the latter aresome proteins that conduct solutes or signals across the membrane.Membrane protein localization has also been investigated withreagents that react with protein side chains but cannot cross membranes (Fig. 10-7). The human erythrocyte is convenient for such studies, because the plasma membrane is the only membrane present.Figure 10-7 Experiments to determine thetransmembrane arrangement of membraneproteins, (a) Both ethylacetimidate (EA) andisoethionylacetimidate (IEA) react with free aminogroups in proteins, but only the ethyl derivative(EA) diffuses freely through the membrane.(b) Comparison of the labeling patterns with thetwo reagents reveals whether a given protein isexposed only on the outer surface or only on theinner surface. Proteins labeled by both reagents,but more heavily by the permeant reagent, are exposed on both sides, and thus span the membrane.Chapter 10 Biological Membranes and TransportBOX 10-1275Electron Microscopy of MembranesCombined with different staining procedures andtissue-preparation methods, electron microscopyhas revealed important details of membrane structure.
Shown here are three different aspects of theerythrocyte membrane, visualized by electron microscopy after three different ways of preparingthe cells for examination.Figure 1 is a transmission electron micrograph of a section through the erythrocyte membrane, showing the two dense lines visible afterosmium tetroxide staining of cells, correspondingto the outer and inner polar layers of the membrane-lipid head groups.
The clear zone betweenthe lines is the hydrophobic portion of the lipid bilayer, which contains the nonpolar fatty acyl tails.Figure 2 shows the glycocalyx on the outer surface of the erythrocyte, visualized by a special$*staining procedure. This "fuzzy coat," which consists of hydrophilic oligosaccharide groups of membrane glycoproteins and glycolipids, is over 100 nmthick, more than ten times the thickness of thelipid bilayer itself.A view of the inside of the erythrocyte membrane (illustrated in Figure 3), is produced by thefreeze-fracture method. In this procedure thecells are frozen and the frozen block is shattered orsplit.
The fracture lines sometimes split a membrane along a plane between the two lipid layers.The exposed surface is coated with a very thinlayer of carbon, then visualized with the electronmicroscope (Fig. 4). The inside surface of one lipidlayer forms the smooth background; the clusters ofglobular bodies are molecules of integral membrane proteins.Outer faceGlycolipidPhospholipidInner faceFigure 1Figure 3Figure 4^GlycoproteinProtein276Part II Structure and CatalysisExperiments like those described in Figure 10-7 show that theglycophorin molecule spans the erythrocyte membrane.
Its aminoterminal domain (bearing the carbohydrate) is on the outer surface ofthe erythrocyte, and its carboxyl terminus protrudes on the insideof the cell. The amino-terminal and carboxyl-terminal domains contain many polar or charged amino acid residues, and are thereforequite hydrophilic. Hov/ever, a long segment in the center of the proteincontains mainly hydrophobic amino acid residues.
These findingssuggest the transmembrane arrangement of glycophorin shown inFigure 10-8.Figure 10-8 Transbilayer disposition ofglycophorin in the erythrocyte. One hydrophilicdomain, containing all the sugar residues, is on theouter surface, and another hydrophilic domain protrudes on the inner surface. The red hexagons represent a tetrasaccharide (containing two NeuNAc,Gal, and GalNAc) O-linked to a serine or threonineresidue; the blue hexagon represents an oligosaccharide chain N-linked to an asparagine residue.
Asegment of relatively hydrophobic residues forms ana helix that traverses the membrane bilayer.AminoterminusCarboxyl (jGln AsP SerterminusMembrane Proteins Are Oriented AsymmetricallyOne further fact may be deduced from the results of the experimentswith glycophorin: it does not move from one face of the bilayer to theother; its disposition in the membrane is asymmetric. Similar studiesof other membrane proteins show that each has a specific orientationin the bilayer, and that protein reorientation byflip-flopdiffusion occurs seldom, if ever. Furthermore, glycoproteins of the plasma membrane are invariably situated with their sugar residues on the outersurface of the cell.
As we shall see, the asymmetric arrangement ofmembrane proteins results in functional asymmetry; all the moleculesof a given ion pump, for example, have the same orientation and therefore all pump in the same direction.PeripheralproteinIntegral Membrane Proteins Are Insoluble in WaterMembrane proteins may be divided operationally into two groups: integral (intrinsic) proteins, which are very firmly bound to the membrane, and peripheral (extrinsic) proteins, which are bound moreloosely, or reversibly.
Peripheral membrane proteins can be releasedfrom membranes by relatively mild treatments (Fig. 10-9), and oncereleased from the membrane they are generally water soluble. In contrast, the release of integral proteins from membranes requires theaction of agents (detergents, organic solvents, or denaturants) that interfere with hydrophobic interactions. Even after integral proteinshave been solubilized, removal of the detergent may cause the proteinto precipitate as an insoluble aggregate. The insolubility of integralmembrane proteins results from the presence of domains rich in hydrophobic amino acids; hydrophobic interactions between the protein andthe lipids of the membrane account for the firm attachment of theprotein.change in pH;chelating agent; \ureaLipid-linkedperipheral protein^Some Integral Proteins Have HydrophobicTransmembrane AnchorsIntegral membrane proteins generally have domains rich in hydrophobic amino acids.
In some proteins, there is a single hydrophobic sequence in the middle of the protein (as in glycophorin) or at the aminoor carboxyl terminus. Other membrane proteins have multiple hydrophobic sequences, each long enough to span the lipid bilayer when inthe a-helical conformation.One of the best-studied membrane-spanning proteins, bacteriorhodopsin, contains seven very hydrophobic internal sequences, andcrosses the lipid bilayer seven times. Bacteriorhodopsin is a lightdriven proton pump that is densely packed in regular arrays in thepurple membrane of the bacterium Halobacterium halobium.
Whenthese arrays are viewed with the electron microscope from several angles, the resulting images allow a three-dimensional reconstruction ofthe bacteriorhodopsin molecule (Fig. 10-10). Seven a-helical segments,each traversing the lipid bilayer, are connected by nonhelical loops atthe inner or outer face of the membrane.
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